Identification and Characterization of the Host Protein DNAJC14 as a Broadly Active Flavivirus Replication Modulator

Viruses in the Flavivirus genus of the Flaviviridae family are arthropod-transmitted and contribute to staggering numbers of human infections and significant deaths annually across the globe. To identify cellular factors with antiviral activity against flaviviruses, we screened a cDNA library using an iterative approach. We identified a mammalian Hsp40 chaperone protein (DNAJC14) that when overexpressed was able to mediate protection from yellow fever virus (YFV)-induced cell death. Further studies revealed that DNAJC14 inhibits YFV at the step of viral RNA replication. Since replication of bovine viral diarrhea virus (BVDV), a member of the related Pestivirus genus, is also known to be modulated by DNAJC14, we tested the effect of this host factor on diverse Flaviviridae family members. Flaviviruses, including the pathogenic Asibi strain of YFV, Kunjin, and tick-borne Langat virus, as well as a Hepacivirus, hepatitis C virus (HCV), all were inhibited by overexpression of DNAJC14. Mutagenesis showed that both the J-domain and the C-terminal domain, which mediates self-interaction, are required for anti-YFV activity. We found that DNAJC14 does not block YFV nor HCV NS2-3 cleavage, and using non-inhibitory mutants demonstrate that DNAJC14 is recruited to YFV replication complexes. Immunofluorescence analysis demonstrated that endogenous DNAJC14 rearranges during infection and is found in replication complexes identified by dsRNA staining. Interestingly, silencing of endogenous DNAJC14 results in impaired YFV replication suggesting a requirement for DNAJC14 in YFV replication complex assembly. Finally, the antiviral activity of overexpressed DNAJC14 occurs in a time- and dose-dependent manner. DNAJC14 overexpression may disrupt the proper stoichiometry resulting in inhibition, which can be overcome upon restoration of the optimal ratios due to the accumulation of viral nonstructural proteins. Our findings, together with previously published work, suggest that the members of the Flaviviridae family have evolved in unique and important ways to interact with this host Hsp40 chaperone molecule

Influenza Virus Susceptibility of Wild-Derived CAST/EiJ Mice Results from Two Amino Acid Changes in the MX1 Restriction Factor

The interferon-regulated Mx1 gene of the A2G mouse strain confers a high degree of resistance against influenza A and Thogoto viruses. Most other laboratory inbred mouse strains carry truncated nonfunctional Mx1 alleles and, consequently, exhibit high virus susceptibility. Interestingly, CAST/EiJ mice, derived from wild Mus musculus castaneus, possess a seemingly intact Mx1 gene but are highly susceptible to influenza A virus challenge. To determine whether the enhanced influenza virus susceptibility is due to intrinsically reduced antiviral activity of the CAST-derived Mx1 allele, we generated a congenic C57BL/6J mouse line that carries the Mx locus of CAST/EiJ mice. Adult animals of this line were almost as susceptible to influenza virus challenge as standard C57BL/6J mice lacking functional Mx1 alleles but exhibited far more pronounced resistance to Thogoto virus. Sequencing revealed that CAST-derived MX1 differs from A2G-derived MX1 by two amino acids (G83R and A222V) in the GTPase domain. Especially the A222V mutation reduced GTPase activity of purified MX1 and diminished the inhibitory effect of MX1 in influenza A virus polymerase activity assays. Further, MX1 protein was substantially less abundant in organs of interferon-treated mice carrying the CAST Mx1 allele than in those of mice carrying the A2G Mx1 allele. We found that the CAST-specific mutations reduced the metabolic stability of the MX1 protein although Mx1 mRNA levels were unchanged. Thus, the enhanced influenza virus susceptibility of CAST/EiJ mice can be explained by minor alterations in the MX1 restriction factor that negatively affect its enzymatic activity and reduce its half-life. IMPORTANCE Although the crystal structure of the prototypic human MXA protein is known, the importance of specific protein domains for antiviral activity is still incompletely understood. Novel insights might come from studying naturally occurring MX protein variants with altered antiviral activity. Here we identified two seemingly minor amino acid changes in the GTPase domain that negatively affect the enzymatic activity and metabolic stability of murine MX1 and thus dramatically reduce the influenza virus resistance of the respective mouse inbred strain. These observations highlight our current inability to predict the biological consequences of previously uncharacterized MX mutations in mice. Since this is probably also true for naturally occurring mutations in Mx genes of humans, careful experimental analysis of any natural MXA variants for altered activity is necessary in order to assess possible consequences of such mutations on innate antiviral immunity

A high-resolution anatomical atlas of the transcriptome in the mouse embryo.

Ascertaining when and where genes are expressed is of crucial importance to understanding or predicting the physiological role of genes and proteins and how they interact to form the complex networks that underlie organ development and function. It is, therefore, crucial to determine on a genome-wide level, the spatio-temporal gene expression profiles at cellular resolution. This information is provided by colorimetric RNA in situ hybridization that can elucidate expression of genes in their native context and does so at cellular resolution. We generated what is to our knowledge the first genome-wide transcriptome atlas by RNA in situ hybridization of an entire mammalian organism, the developing mouse at embryonic day 14.5. This digital transcriptome atlas, the Eurexpress atlas (http://www.eurexpress.org), consists of a searchable database of annotated images that can be interactively viewed. We generated anatomy-based expression profiles for over 18,000 coding genes and over 400 microRNAs. We identified 1,002 tissue-specific genes that are a source of novel tissue-specific markers for 37 different anatomical structures. The quality and the resolution of the data revealed novel molecular domains for several developing structures, such as the telencephalon, a novel organization for the hypothalamus, and insight on the Wnt network involved in renal epithelial differentiation during kidney development. The digital transcriptome atlas is a powerful resource to determine co-expression of genes, to identify cell populations and lineages, and to identify functional associations between genes relevant to development and disease

In vivo evasion of MxA by avian influenza viruses requires human signature in the viral nucleoprotein

Zoonotic transmission of influenza A viruses can give rise to devastating pandemics, but currently it is impossible to predict the pandemic potential of circulating avian influenza viruses. Here, we describe a new mouse model suitable for such risk assessment, based on the observation that the innate restriction factor MxA represents an effective species barrier that must be overcome by zoonotic viruses. Our mouse lacks functional endogenous Mx genes but instead carries the human MX1 locus as a transgene. Such transgenic mice were largely resistant to highly pathogenic avian H5 and H7 influenza A viruses, but were almost as susceptible to infection with influenza viruses of human origin as nontransgenic littermates. Influenza A viruses that successfully established stable lineages in humans have acquired adaptive mutations which allow partial MxA escape. Accordingly, an engineered avian H7N7 influenza virus carrying a nucleoprotein with signature mutations typically found in human virus isolates was more virulent in transgenic mice than parental virus, demonstrating that a few amino acid changes in the viral target protein can mediate escape from MxA restriction in vivo. Similar mutations probably need to be acquired by emerging influenza A viruses before they can spread in the human population.</jats:p

Proposed model of DNAJC14 function.

<p>(A) Incoming viral RNA is translated to produce the inactive form(s) of a viral protein(s) required for RNA replication complex formation (A and B, yellow and brown stars). When the stoichiometry of the substrate protein(s) and the host chaperone machinery, consisting of DNAJC14 (DNAJ, blue) and Hsp70 (green), is appropriate, proper folding (AB) allows the formation of replication complexes which generate new progeny viral RNA. This RNA is further translated to produce more substrate, which after undergoing the chaperone process results in the formation of additional replication complexes and amplification of the RNA replication process. Overexpression of DNAJC14 mutants (red) lacking the C-terminal self-interaction domain (CT mutants), or with mutations in the J domain (H471Q, L466P) has no effect on viral replication, since the mutants lack features necessary for stable interaction and the chaperone complex is not disrupted. (B) Overexpression of full-length DNAJC14 (FL) or N-terminal truncation mutants (NT mutants) results in their incorporation into the chaperone complexes due to the presence of the C-terminal interaction domain and an intact J domain. Disruption of the normal stoichiometry of the substrate/chaperone complex results in a failure to properly fold the viral protein and a failure to generate replication complexes. With time, however, continued translation of the incoming genome results in increased concentrations of the viral protein. The appropriate stoichiometry is restored allowing the chaperone process to proceed and viral replication complexes to be generated.</p

DNAJC14 inhibits YFV in a temporal and dose-dependent manner.

<p>(A) SW13 cells were mock transduced (Mock) or transduced with Trip-RFP-hNZAP (NZAP), Trip-RFP-hDNAJC14-FL (FL) or Trip-RFP-hDNAJC14-NT1 (NT1) and infected 2 d later with YFV-Venus (moi = 5). Cells were fixed at 1.5 d post infection and analyzed by flow cytometry. The Venus (y-axis) and RFP (x-axis) fluorescence intensities of the cells are shown; gates to indicate expression of the transduced protein or productive infection were set on Mock transduced, uninfected cells (not shown). (B) SW13 cells were transduced with Trip-RFP-hDNAJC14-NT1 and infected 2 d later with YFV-Venus (moi = 5) and analyzed as in (A) at the indicated days after infection. (C) SW13 cells were left untransduced (closed circles) or were transduced with Trip-RFP-hDNAJC14-NT1 (RFP-NT1, open circles) and infected 2 d later with YFV-Venus (moi = 5). At the indicated times, the medium was removed and YFV present in the medium was quantified by plaque assay. Each data point represents the mean titer obtained from duplicate wells; error bars indicating the range are obscured by the symbols. The dotted line indicates the sensitivity of the plaque assay.</p